Measuring aptamer equilibria using gradient micro free flow electrophoresis

Ryan T. Turgeon, Bryan R. Fonslow, Meng Jing, Michael T. Bowser

Research output: Contribution to journalArticle

36 Scopus citations

Abstract

Gradient micro free flow electrophoresis (μFFE) was used to observe the equilibria of DNA aptamers with their targets (IgE or HIVRT) across a range of ligand concentrations. A continuous stream of aptamer was mixed online with an increasing concentration of target and introduced into the μFFE device, which separated ligand-aptamer complexes from the unbound aptamer. The continuous nature of μFFE allowed the equilibrium distribution of aptamer and complex to be measured at 300 discrete target concentrations within 5 min. This is a significant improvement in speed and precision over affinity capillary electrophoresis (ACE) assays. The dissociation constant of the aptamer-IgE complex was estimated to be 48 ± 3 nM. The high coverage across the range of ligand concentrations allowed complex stoichiometries of the aptamer-HIVRT complexes to be observed. Nearly continuous observation of the equilibrium distribution from 0 to 500 nM HIVRT revealed the presence of complexes with 3:1 (aptamer/HIVRT), 2:1, and 1:1 stoichiometries.

Original languageEnglish (US)
Pages (from-to)3636-3641
Number of pages6
JournalAnalytical chemistry
Volume82
Issue number9
DOIs
StatePublished - May 1 2010

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